PROJECT SUMMARY/ABSTRACT Marijuana is one of the most widely used drugs of abuse and is increasingly being ascribed therapeutic properties. Unfortunately, the structure of the ?marijuana receptor?, CB1, is still not known. Thus, the mechanisms through which drugs bind and activate CB1, the highest expressed GPCR in the brain, are still unclear at an atomic level. Given the key role this receptor plays in cognition, and its potential as a high-value therapeutic target for treating addiction, chronic pain and glaucoma, why has the structure of CB1 never been determined? On reason is that CB1 has proven to be extremely difficult to purify and manipulate in a functional form. However, after many years of concerted effort, our lab has overcome this obstacle and we can now routinely prepare milligram quantities of purified, functional CB1. Thus, in this R21 we now propose to solve the structure of CB1 using two complementary approaches. The first will involve crystallography in collaboration with Dr. Brian Kobilka, our long-term collaborator and world expert in solving GPCR structures. The second will use single particle EM analysis, a rapidly advancing method that is dramatically changing the face of structural biology. The work will be done with Dr. James Chen, an expert in solving protein structures using EM, and my next-door colleague. Two Specific Aims are proposed. Specific Aim 1 (SA1) will employ several approaches to crystalize CB1, leveraging our expertise in working with this receptor and the expertise of Dr. Kobilka. These include using CB1 chimeras and CB1 mutants that we have identified to increase receptor expression and stability. In Specific Aim 2 (SA2), we will use single particle EM analysis to help screen and optimize the crystallization conditions described in SA1, and to determine the structure of CB1 complexed with itself and in complex with the G?i subunit. Since single particle EM has does not require crystals, and can be carried out on the protein under more native like, soluble conditions, these EM data will provide unique structural data about how GPCRs dimerize and elucidate how they interact with G?i subunits, information still not known for any GPCR. We anticipate our results will be of significant interest to the pharmacological research community, and the wide array of people interested in this intriguing, lipid binding receptor. Understanding of how drugs bind and activate CB1 will ultimately require high-resolution structures, thus we feel the work we are proposing is both inevitable and long overdue. Such information will be critical to enable rational design and fine tuning of ligands that bind and modulate CB1 activity, for example to preferentially induce biased signaling. Our proposed work is also synergistic ? structures obtained from the CB1 crystals will prove invaluable for further refining the single particle EM data. Finally, we note that the methods we develop should be broadly applicable for studying the structure of other challenging GPCRs and their interactions with affiliate proteins.